Research leading to the present invention was supported in part by funds from the National Institutes of Health, the National Cancer Institute, and the Leonard and Madlyn Abramson Family Cancer Research Institute Fund.
Structural changes in proteins can be induced by various physical factors including pH, solvents, ligand binding and oligomerization. Conformational changes can occur at a defined local site or, as observed in multimeric proteins, at a distance from the ligand binding site (allosterism).
Protein function can be altered by conformational changes. Immunoglobulins have been shown to alter the function of proteins by inducing small to large conformational changes, and by affecting the oligomerization of proteins. For example, in the crystal structure of Taq DNA polymerase complex, an antibody inhibited the function of DNA polymerase by inducing a large conformational change in the helix and trapping the protein in a transition state suggesting that altering conformational configuration at distinct sites away from the binding sites might be used to modulate protein function.
It has been generally argued that conformational changes may be a step in substrate/ligand recognition. Several studies from the crystal structures of protein-protein complexes revealed conformational changes ranging from 2-20 Å (0.2-2 nm) either locally or globally between subdomains. In the case of multimeric proteins such as myoglobin or glycogen phosphorylase, with known allosteric sites, defined conformational changes are transmitted through regions of the protein for regulatory or functional effects.
While surface cavities on non-enzymatic classes of proteins have been largely unexplored, inactivation of enzymes has been accomplished by designing competitive or substrate analog inhibitors that bind at active sites. Several therapeutic inhibitors have been developed based on the structure and molecular properties of substrates and these are generally known as “substrate analogs”. Small molecule effectors have been identified for enzymes. For example, allosteric inhibitors have been designed and developed based on the knowledge of known and established allosteric binding sites. Small conformational perturbations near the active site/ligand binding sites or polymorphisms near the active site have been suggested to be responsible for resistance to substrate based inhibitors.
Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine produced by activated macrophages/monocytes and lymphocytes. TNF-α is a potent mediator in inflammatory and immune responses, including the recruitment of leukocytes to injured tissues during bacterial and other microbial infections, and following stimulation with inflammatory substances. When present in excessive quantities, TNF-α is known to cause tissue injury, and has been implicated in the pathology associated with inflammatory and autoimmune diseases.
The biological effects of TNF-α, are mediated through two distinct membrane-protein receptors, TNF-RI and TNF-RII (in humans, p55 and p75, respectively), which differ in sequence and molecular mass. TNF-RI is reported to be present at low levels in most, if not all, human cell types, and expression of the TNF-RI gene in humans can be upregulated by infection, interferons, and modulators of second messengers, such as phorbol esters. The extracellular portions of both TNF receptors also exist in soluble forms, which are derived from membrane-bound forms of the receptors by proteolytic cleavage at the cell surface. The soluble TNF receptors retain the ability to bind TNF-α in solution. Soluble TNF receptors have been identified in urine and sera from healthy individuals, and have been shown to be elevated in some chronic diseases and following inoculation with agents that induce TNF-α release.
The pathological effects of TNF-α can be alleviated by administration of soluble TNF-R fragments or anti-TNF-α antibodies. These agents bind circulating TNF-α, thus preventing the binding of TNF-α to TNF-R and lowering TNF-α signaling. TNF-R fragments or anti-TNF-α antibodies have been approved, by the U.S. Food and Drug Administration, for treatment of rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis.
The efficacy of TNF-R fragments and anti-TNF-α antibodies in treating TNF-α-mediated conditions demonstrates that reducing signaling through the TNF-α/TNF-R signaling pathway can be used effectively to treat TNF-α-mediated conditions. TNF-R fragments and anti-TNF-α antibodies, however, are expensive to produce. Moreover, these proteinaceous agents require intravenous administration.
There is, therefore, a need in the art for additional agents that reduce signaling through the TNF-α/TNF-R signaling pathway and that can be used for treatment of TNF-α-mediated conditions. Accordingly, the present inventors have discovered small molecule compounds that bind to an allosteric site on TNF-R1, thus inhibiting binding of TNF-α to TNF-R1 and reducing activity of the TNF-α/TNF-R1 signaling pathway. The compounds are useful for treatment of TNF-α mediated conditions.